Reference Genes For Transcript Normalization Research Articles

Evaluation of reference genes for transcript normalization in Fragaria chiloensis fruit and vegetative tissues.

The online version contains supplementary material available at 10.1007/s12298-022-01227-y.

Systematic screening and validation of reliable reference genes for qRT-PCR analysis in Okra (Abelmoschus esculentus L.)

Quantitative real-time polymerase chain reaction (qRT-PCR) is a sensitive and widely used technique for quantifying gene expression levels, and its accuracy depends on the reference genes used for data normalization. To date, no reference gene has been reported in the nutritious and functional vegetable okra (Abelmoschus esculentus L.). Herein, 11 candidates of reference genes were selected and evaluated for their expression stability in okra in different tissues at different developmental stages by using three software algorithms (geNorm, NormFinder, BestKeeper) and a web-based tool (RefFinder). Among them, eukaryotic initiation factor 4 alpha (eIF4A) and protein phosphatase 2A (PP2A) showed the highest stability, while TUA5 had the lowest stability. The combined usage of these two most stable reference genes was sufficient to normalize gene expression in okra. Then, the above results were further validated by normalizing the expression of the cellulose synthase gene CesA4. This work provides appropriate reference genes for transcript normalization in okra, which will facilitate subsequent functional gene research on this vegetable crop.

Selection and evaluation of suitable reference genes for quantitative gene expression analysis during infection of Cucumis sativus with Pectobacterium brasiliense.

Bacterial soft rot caused by Pectobacterium brasiliense (Pbr) has resulted in severe economic losses of cucumber production in northern China. Quantitative reverse transcription PCR (RT-qPCR) is widely used to determine the fold change in the expression of genes of interest, and an appropriate reference gene played a critical role in the evaluation of genes expression. However, the suitable reference genes for transcript normalization during the interaction between cucumber and Pbr have not yet been systematically validated. In this study, we aimed to identify the suitable reference genes for accurate and reliable normalization of cucumber and Pbr RT-qPCR data. We selected 14 candidate reference genes for cucumber and 10 candidate reference genes for Pbr were analysed by using four algorithms (the deltaCt method, BestKeeper, NormFinder and geNorm). Furthermore, five genes in cucumber involved in plant resistance and five genes in Pbr related to the virulence were selected to confirm the reliability of the reference genes by RT-qPCR. CsARF (ADP-ribosylation factor 1) and pgi (glucose-6-phosphate isomerase) were suggested as the most suitable reference genes for cucumber and Pbr respectively. Our results suggested that CsARF (ADP-ribosylation factor 1) and pgi (glucose-6-phosphate isomerase) could be as the reference genes to normalize expression data for cucumber and Pbr during the process of pathogen-host interaction respectively. To our knowledge, this is the first systematic study of the optimal reference genes specific to cucumber and Pbr, which could help advance the molecular interactions research in Cucurbitaceae vegetables and Pectobacterium species pathosystems.

Genome-wide identification of ubiquitin proteasome subunits as superior reference genes for transcript normalization during receptacle development in strawberry cultivars

BackgroundGene transcripts that show invariant abundance during development are ideal as reference genes (RGs) for accurate gene expression analyses, such as RNA blot analysis and reverse transcription–quantitative real time PCR (RT-qPCR) analyses. In a genome-wide analysis, we selected three “Commonly used” housekeeping genes (HKGs), fifteen “Traditional” HKGs, and nine novel genes as candidate RGs based on 80 publicly available transcriptome libraries that include data for receptacle development in eight strawberry cultivars.ResultsThe results of the multifaceted assessment consistently revealed that expression of the novel RGs showed greater stability compared with that of the “Commonly used” and “Traditional” HKGs in transcriptome and RT-qPCR analyses. Notably, the majority of stably expressed genes were associated with the ubiquitin proteasome system. Among these, two 26 s proteasome subunits, RPT6A and RPN5A, showed superior expression stability and abundance, and are recommended as the optimal RGs combination for normalization of gene expression during strawberry receptacle development.ConclusionThese findings provide additional useful and reliable RGs as resources for the accurate study of gene expression during receptacle development in strawberry cultivars.

Genome-wide identification and testing of superior reference genes for transcript normalization during analyses of flesh development in Asian pear cultivars

Quantitative real-time polymerase chain reaction (qRT-PCR) is currently the best available option for analyzing gene expression and transcriptomes. In this context, it is important to use ideal reference genes (RGs) to normalize the expression data for the samples to be compared. Thus, the present study was established to identify and validate superior RGs for qRT-PCR analyses of fruit development in Asian pear cultivars. A total of seven “commonly used” RGs, seven “traditional” housekeeping genes (HKGs), and four novel genes were selected as candidate RGs based on 35 publicly available transcriptome libraries that include data for fruit development in five Asian pear cultivars. Transcriptomic and qRT-PCR analyses consistently revealed that the novel RGs were expressed more stably than “commonly used” RGs and “traditional” HKGs. Among the novel RGs, BPS1 and ICDH1 were the most highly and stably expressed genes. Moreover, these two genes formed the optimal RG combination for normalizing gene expression data during analyses of pear flesh development. These findings may help researchers optimize their selection of RGs for future investigations of gene expression during flesh development in Asian pear cultivars.

Identification of reference genes for transcript normalization in various tissue types and seedlings subjected to different abiotic stresses of woodland strawberry Fragaria vesca

Quantitative real-time PCR (qRT-PCR) is one of the most popular methods for gene expression studies. However, data on the usage of reference genes in strawberry are limited. In this study, we assayed expression dynamics of 20 candidate reference genes across various organs (root, leaf and flower), different developmental stages of fruits, and in seedlings under abiotic stresses (heat, cold, drought and salt) in woodland strawberry Fragaria vesca. Expression stability of those genes was analyzed by four commonly used statistical algorithms (geNorm, NormFinder, BestKeeper and dalteCt) for comparisons and rankings. Our results indicate that the expression stability of reference genes varies according to tissue types and experimental conditions. Furthermore, two reference genes are sufficient for normalization in tissues and seedlings under the four stress conditions in Fragaria vesca. We recommend FveSAND and FveMT1, FveCHC1 and FveGAPDH as reference genes in organs and fruits, FveCHC1 and FveTIM1, FveSAND and FveTIM1, FveCHP1 and FveAP1, FveCHC1 and FveEF 1a under heat, cold, drought and salt stress conditions, respectively. Our study provides a set of reliable standard reference genes for expression analyses of genes in various tissue types and seedlings under different environment conditions of Fragaria vesca.

Selection of reference genes for qPCR normalization in buffalobur (Solanum rostratum Dunal)

Buffalobur (Solanum rostratum Dunal), which belongs to the Solanaceae family, is a worldwide noxious invasive weed and is listed as one of the top 10 alien invasive species in China. It is harmful to humans and livestock because the entire plant is covered with spines containing toxins. Many studies have analysed the gene expression in this weed species under different stress conditions using quantitative real-time PCR (qPCR). However, until now, there has been no report on suitable reference genes in buffalobur. Herein, 14 candidate reference genes were selected and evaluated for their expression stability in buffalobur in different tissues, at different developmental stages, and in response to several stress conditions using the geNorm, NormFinder, BestKeeper and RefFinder statistical algorithms. The results showed that EF1α, ACT and SAND are suitable reference genes across all samples tested. We recommend the normalization of target gene expression under different experimental conditions using these three genes together. Validation of selected reference genes was achieved by assessing the relative expression levels of P5CS and GI. This work identified the appropriate reference genes for transcript normalization in buffalobur, which will be helpful in future genetic studies of this invasive weed.

Selection of Reference Genes for Real-time Quantitative PCR Normalization in the Process of Gaeumannomyces graminis var. tritici Infecting Wheat.

Gaeumannomyces graminis var. tritici is a soil borne pathogenic fungus associated with wheat roots. The accurate quantification of gene expression during the process of infection might be helpful to understand the pathogenic molecular mechanism. However, this method requires suitable reference genes for transcript normalization. In this study, nine candidate reference genes were chosen, and the specificity of the primers were investigated by melting curves of PCR products. The expression stability of these nine candidates was determined with three programs-geNorm, Norm Finder, and Best Keeper. TUBβ was identified as the most stable reference gene. Furthermore, the exopolygalacturonase gene (ExoPG) was selected to verify the reliability of TUBβ expression. The expression profile of ExoPG assessed using TUBβ agreed with the results of digital gene expression analysis by RNA-Seq. This study is the first systematic exploration of the optimal reference genes in the infection process of Gaeumannomyces graminis var. tritici.

Genome-wide identification and validation of new reference genes for transcript normalization in developmental and post-harvested fruits of Actinidia chinensis

The appropriate reference genes are important and essential for reliable results of transcript normalization in real-time qRT-PCR. In the current study, we identified 1203 stably expressed genes from 35,286 genes' expression profiles in developmental fruits of Actinidia chinensis. We manually selected six candidate genes and assessed their expression levels, using two sets of fruit samples of A. chinensis: flesh fruits at four developmental stages and post-harvested fruits. The expression stability of these six genes was assessed by three independent algorithms: geNorm, NormFinder, and BestKeeper. Statistical results indicated these six genes can serve as internal control in both developmental and post-harvested fruits. Among these genes, UBQ_CONJ_E2 (Ubiquitin-conjugating enzyme E2 36) and TUB_FCB (Tubulin folding cofactor B) were the two best reference genes identified in this study. The identification and validation of these reference genes can be helpful for elucidating the studies of fruit development and post-harvested fruits' storage in A. chinensis and other fruit crops of Actinidiaceae.

Reference Gene Selection for qRT-PCR Normalization Analysis in kenaf (Hibiscus cannabinus L.) under Abiotic Stress and Hormonal Stimuli.

Kenaf (Hibiscus cannabinus L.), an environmental friendly and economic fiber crop, has a certain tolerance to abiotic stresses. Identification of reliable reference genes for transcript normalization of stress responsive genes expression by quantitative real-time PCR (qRT-PCR) is important for exploring the molecular mechanisms of plants response to abiotic stresses. In this study, nine candidate reference genes were cloned, and their expression stabilities were assessed in 132 abiotic stress and hormonal stimuli samples of kenaf using geNorm, NormFinder, and BestKeeper algorithms. Results revealed that HcPP2A (Protein phosphatase 2A) and HcACT7 (Actin 7) were the optimum reference genes across all samples; HcUBC (Ubiquitin-conjugating enzyme like protein) was the worst reference gene for transcript normalization. The reliability of the selected reference genes was further confirmed by evaluating the expression profile of HcWRKY28 gene at different stress durations. This work will benefit future studies on discovery of stress-tolerance genes and stress-signaling pathways in this important fiber crop.

Selection of relatively exact reference genes for gene expression studies in goosegrass (Eleusine indica) under herbicide stress

Goosegrass (Eleusine indica) is one of the most serious annual grassy weeds worldwide, and its evolved herbicide-resistant populations are more difficult to control. Quantitative real-time PCR (qPCR) is a common technique for investigating the resistance mechanism; however, there is as yet no report on the systematic selection of stable reference genes for goosegrass. This study proposed to test the expression stability of 9 candidate reference genes in goosegrass in different tissues and developmental stages and under stress from three types of herbicide. The results show that for different developmental stages and organs (control), eukaryotic initiation factor 4 A (eIF-4) is the most stable reference gene. Chloroplast acetolactate synthase (ALS) is the most stable reference gene under glyphosate stress. Under glufosinate stress, eIF-4 is the best reference gene. Ubiquitin-conjugating enzyme (UCE) is the most stable reference gene under quizalofop-p-ethyl stress. The gene eIF-4 is the recommended reference gene for goosegrass under the stress of all three herbicides. Moreover, pairwise analysis showed that seven reference genes were sufficient to normalize the gene expression data under three herbicides treatment. This study provides a list of reliable reference genes for transcript normalization in goosegrass, which will facilitate resistance mechanism studies in this weed species.

Selection of reliable reference genes for RT-qPCR analysis during developmental stages and abiotic stress in Setaria viridis.

Real-time PCR (RT-qPCR) expression analysis is a powerful analytical technique, but reliable results depend on the use of stable reference genes for proper normalization. This study proposed to test the expression stability of 13 candidate reference genes in Setaria viridis, a monocot species recently proposed as a new C4 model plant. Gene expression stability of these genes was assayed across different tissues and developmental stages of Setaria and under drought or aluminum stress. In general, our results showed Protein Kinase, RNA Binding Protein and SDH as the most stable genes. Moreover, pairwise analysis showed that two reference genes were sufficient to normalize the gene expression data under each condition. By contrast, GAPDH and ACT were the least stably expressed genes tested. Validation of suitable reference genes was carried out to profile the expression of P5CS and GolS during abiotic stress. In addition, normalization of gene expression of SuSy, involved in sugar metabolism, was assayed in the developmental dataset. This study provides a list of reliable reference genes for transcript normalization in S. viridis in different tissues and stages of development and under abiotic stresses, which will facilitate genetic studies in this monocot model plant.

Evaluation of Appropriate Reference Genes for Gene Expression Normalization during Watermelon Fruit Development.

Gene expression analysis in watermelon (Citrullus lanatus) fruit has drawn considerable attention with the availability of genome sequences to understand the regulatory mechanism of fruit development and to improve its quality. Real-time quantitative reverse-transcription PCR (qRT-PCR) is a routine technique for gene expression analysis. However, appropriate reference genes for transcript normalization in watermelon fruits have not been well characterized. The aim of this study was to evaluate the appropriateness of 12 genes for their potential use as reference genes in watermelon fruits. Expression variations of these genes were measured in 48 samples obtained from 12 successive developmental stages of parthenocarpic and fertilized fruits of two watermelon genotypes by using qRT-PCR analysis. Considering the effects of genotype, fruit setting method, and developmental stage, geNorm determined clathrin adaptor complex subunit (ClCAC), β-actin (ClACT), and alpha tubulin 5 (ClTUA5) as the multiple reference genes in watermelon fruit. Furthermore, ClCAC alone or together with SAND family protein (ClSAND) was ranked as the single or two best reference genes by NormFinder. By using the top-ranked reference genes to normalize the transcript abundance of phytoene synthase (ClPSY1), a good correlation between lycopene accumulation and ClPSY1 expression pattern was observed in ripening watermelon fruit. These validated reference genes will facilitate the accurate measurement of gene expression in the studies on watermelon fruit biology.

Selection and validation of reference genes for transcript normalization in gene expression studies in Catharanthus roseus

Quantitative Real-Time PCR (qPCR), a sensitive and commonly used technique for gene expression analysis, requires stably expressed reference genes for normalization of gene expression. Up to now, only one reference gene for qPCR analysis, corresponding to 40S Ribosomal protein S9 (RPS9), was available for the medicinal plant Catharanthus roseus, the only source of the commercial anticancer drugs vinblastine and vincristine. Here, we screened for additional reference genes for this plant species by mining C. roseus RNA-Seq data for orthologs of 22 genes known to be stably expressed in Arabidopsis thaliana and qualified as superior reference genes for this model plant species. Based on this, eight candidate C. roseus reference genes were identified and, together with RPS9, evaluated by performing qPCR on a series of different C. roseus explants and tissue cultures. NormFinder, geNorm and BestKeeper analyses of the resulting qPCR data revealed that the orthologs of At2g28390 (SAND family protein, SAND), At2g32170 (N2227-like family protein, N2227) and At4g26410 (Expressed protein, EXP) had the highest expression stability across the different C. roseus samples and are superior as reference genes as compared to the traditionally used RPS9. Analysis of publicly available C. roseus RNA-Seq data confirmed the expression stability of SAND and N2227, underscoring their value as reference genes for C. roseus qPCR analysis.

Selection and Validation of Reference Genes for Real-Time Quantitative PCR in Hyperaccumulating Ecotype of Sedum alfredii under Different Heavy Metals Stresses

Real-time Quantitative PCR (RT-qPCR) has become an effective method for accurate analysis of gene expression in several biological systems as well as under different experimental conditions. Although with high sensitivity, specificity and broad dynamic range, this method requires suitable reference genes for transcript normalization in order to guarantee reproducible and meaningful results. In the present study, we evaluated five traditional housekeeping genes and five novel reference genes in Hyperaccumulating ecotype of Sedum alfredii, a well known hyperaccumulator for heavy metals phytoremediation, under Cd, Pb, Zn and Cu stresses of seven different durations. The expression stability of these ten candidates were determined with three programs - geNorm, NormFinder and BestKeeper. The results showed that all the selected reference genes except for SAND could be used for RT-qPCR normalization. Among them UBC9 and TUB were ranked as the most stable candidates across all samples by three programs together. For the least stable reference genes, however, BestKeeper produced different results compared with geNorm and NormFinder. Meanwhile, the expression profiles of PCS under Cd, Pb, Zn and Cu stresses were assessed using UBC9 and TUB respectively, and similar trends were obtained from the results of the two groups. The distinct expression patterns of PCS indicated that various strategies could be taken by plants in adaption to different heavy metals stresses. This study will provide appropriate reference genes for further gene expression quantification using RT-qPCR in Hyperaccumulator S. alfredii.

Identification and validation of reference genes for transcript normalization in strawberry (Fragaria × ananassa) defense responses.

Strawberry (Fragaria spp) is an emerging model for the development of basic genomics and recombinant DNA studies among rosaceous crops. Functional genomic and molecular studies involve relative quantification of gene expression under experimental conditions of interest. Accuracy and reliability are dependent upon the choice of an optimal reference control transcript. There is no information available on validated endogenous reference genes for use in studies testing strawberry-pathogen interactions. Thirteen potential pre-selected strawberry reference genes were tested against different tissues, strawberry cultivars, biotic stresses, ripening and senescent conditions, and SA/JA treatments. Evaluation of reference candidate’s suitability was analyzed by five different methodologies, and information was merged to identify best reference transcripts. A combination of all five methods was used for selective classification of reference genes. The resulting superior reference genes, FaRIB413, FaACTIN, FaEF1α and FaGAPDH2 are strongly recommended as control genes for relative quantification of gene expression in strawberry. This report constitutes the first systematic study to identify and validate optimal reference genes for accurate normalization of gene expression in strawberry plant defense response studies.

Reference genes for accurate transcript normalization in citrus genotypes under different experimental conditions.

Real-time reverse transcription PCR (RT-qPCR) has emerged as an accurate and widely used technique for expression profiling of selected genes. However, obtaining reliable measurements depends on the selection of appropriate reference genes for gene expression normalization. The aim of this work was to assess the expression stability of 15 candidate genes to determine which set of reference genes is best suited for transcript normalization in citrus in different tissues and organs and leaves challenged with five pathogens (Alternaria alternata, Phytophthora parasitica, Xylella fastidiosa and Candidatus Liberibacter asiaticus). We tested traditional genes used for transcript normalization in citrus and orthologs of Arabidopsis thaliana genes described as superior reference genes based on transcriptome data. geNorm and NormFinder algorithms were used to find the best reference genes to normalize all samples and conditions tested. Additionally, each biotic stress was individually analyzed by geNorm. In general, FBOX (encoding a member of the F-box family) and GAPC2 (GAPDH) was the most stable candidate gene set assessed under the different conditions and subsets tested, while CYP (cyclophilin), TUB (tubulin) and CtP (cathepsin) were the least stably expressed genes found. Validation of the best suitable reference genes for normalizing the expression level of the WRKY70 transcription factor in leaves infected with Candidatus Liberibacter asiaticus showed that arbitrary use of reference genes without previous testing could lead to misinterpretation of data. Our results revealed FBOX, SAND (a SAND family protein), GAPC2 and UPL7 (ubiquitin protein ligase 7) to be superior reference genes, and we recommend their use in studies of gene expression in citrus species and relatives. This work constitutes the first systematic analysis for the selection of superior reference genes for transcript normalization in different citrus organs and under biotic stress.

Genome-Wide Identification and Testing of Superior Reference Genes for Transcript Normalization in Arabidopsis

Gene transcripts with invariant abundance during development and in the face of environmental stimuli are essential reference points for accurate gene expression analyses, such as RNA gel-blot analysis or quantitative reverse transcription-polymerase chain reaction (PCR). An exceptionally large set of data from Affymetrix ATH1 whole-genome GeneChip studies provided the means to identify a new generation of reference genes with very stable expression levels in the model plant species Arabidopsis (Arabidopsis thaliana). Hundreds of Arabidopsis genes were found that outperform traditional reference genes in terms of expression stability throughout development and under a range of environmental conditions. Most of these were expressed at much lower levels than traditional reference genes, making them very suitable for normalization of gene expression over a wide range of transcript levels. Specific and efficient primers were developed for 22 genes and tested on a diverse set of 20 cDNA samples. Quantitative reverse transcription-PCR confirmed superior expression stability and lower absolute expression levels for many of these genes, including genes encoding a protein phosphatase 2A subunit, a coatomer subunit, and an ubiquitin-conjugating enzyme. The developed PCR primers or hybridization probes for the novel reference genes will enable better normalization and quantification of transcript levels in Arabidopsis in the future.